1990 Kluwer Academic Publishers . Printed in Belgium . Trophic interactions among heterotrophic microplankton, nanoplankton, and bacteria in Lake Constance.
Hydrobiologia 191 : 111-122, 1990. P. Biro and J. F. Tailing (eds), Trophic Relationships in Inland Waters . © 1990 Kluwer Academic Publishers . Printed in Belgium .
111
Trophic interactions among heterotrophic microplankton, nanoplankton, and bacteria in Lake Constance Thomas Weisse Limnological Institute, University
of Konstanz, P .O. Box 5560, D-7750 Konstanz, F.R .G.
Key words : microbial loop, HNF, ciliates, dilution experiments, Lake Constance
Abstract A considerable portion of the pelagic energy flow in Lake Constance (FRG) is channelled through a highly dynamic microbial food web . In-situ experiments using the lake water dilution technique according to Landry & Hasset (1982) revealed that grazing by heterotrophic nanoflagellates (HNF) smaller than 10 im is the major loss factor of bacterial production . An average flagellate ingests 10 to 100 bacteria per hour . Nano- and micro-ciliates have been identified as the main predators of HNF . If no other food is used between 3 and 40 HNF are consumed per ciliate and hour . Other protozoans and small metazoans such as rotifers are of minor importance in controlling HNF population dynamics . Clearance rates varied between 0 .2 and 122 .8 nl HNF - ' h - ' and between 0 .2 and 53 .6111 ciliate -' h -', respectively . Ingestion and clearance rates measured for HNF and ciliates are in good agreement with results obtained by other investigators from different aquatic environments and from laboratory cultures . Both the abundance of all three major microheterotrophic categories - bacteria, HNF, and ciliates - and the grazing pressure within the microbial loop show pronounced seasonal variations .
Introduction It has now widely been accepted that a highly dynamic `microbial loop' (Azam et al., 1983) consisting of pelagic bacteria, autotrophic pico- and nanoplankton, heterotrophic nanoflagellates, and microciliates is an integral part of the planktonic food web (for reviews, see Pomeroy, 1974, 1984 ; Williams, 1981 ; Porter etal., 1985 ; E .B . Sherr et al., 1986). A number of recent investigations from different marine areas has shown that heterotrophic nanoflagellates (HNF) are the major consumers of free-living pelagic bacteria (summarized by Sherr & Sherr, 1984) . Small aloricate ciliates (E . B .
Sherr et al., 1986 ; Sherr et al., 1989) and chloroplast-containing chrysomonads (Bird & Kalff, 1986, 1987 ; Estep et al., 1986) have also been implicated as potentially significant bacterial grazers . The fate of HNF and other protozoan production, however, is virtually unknown . Thus, the issue whether the microbial loop forms a `link' or a `sink' of carbon and energy flow along the planktonic food web cannot be answered at present (E . B . Sherr et al., 1986). In freshwater habitats our present knowledge of the microbial loop is extremely scarce . Freshwater species of HNF and microciliates have been cultured on a bacterial diet in the laboratory (for review see Sherr & Sherr, 1984) . There is also
1 12 some evidence that HNF grazing may control the population dynamics of freshwater pelagic bacteria (Gtide, 1986 ; Sanders & Porter, 1986 ; Bloem & Bar-Gilissen, 1989 ; Bloem et ai., 1989) . However, studies in situ on grazing of freshwater HNF are still lacking . The same holds true for the grazing impact of freshwater ciliates on bacteria and HNF . This paper presents results of experiments in situ on growth and grazing of pelagic bacteria, HNF, and microciliates in Lake Constance (FRG) . The trophic relationships among the principal members of the microbial loop will be discussed and compared with results obtained in similar marine studies . The present investigation is part of an extended study on the carbon flow in Lake Constance within the 'Sonderforschungsbereich 248' .
Material and methods Integral samples covering the entire water column from 0 to 6 m were taken with a 4-liter volume, 2-meter long water sampler (developed by the Limnological Institute Constance) at the deepest site of `Oberlinger See', the north western bay of Lake Constance . Samples were taken on 23 occasions between 15 September 1986 and 11 August 1987 at weekly to biweekly intervals . In January and February 1987 no sampling was conducted for technical reasons . To estimate growth rates and grazing pressure within the microbial community, experiments were conducted in situ using a dilution technique and analysis bags (modified after Landry & Hasset, 1982) . Natural samples were pre-filtered through 200-µm-meshed gauze to exclude larger zooplankton, poured into a prerinsed 30-liter volume plastic tub and carefully mixed . Half of the water was sterilized by filtering through membrane filters of 0 .2 µm pore size . The remaining water containing natural microplankton assemblages was then combined with the filtered water in ratios 1 :0, 1 : 1, and 1 :2. Each dilution mixture was dispensed in triplicate into 1-liter volume dialysis sacs (Spetrapor 2, 12000 MW cutoff) . The sacs were individually
placed into coarse synthetic fibre mesh bags, tied into a plastic frame, and the whole device connected to a surface buoy so that the dialysis sacs were suspended 3-3 .5 m below the water surface . In situ incubation lasted for 24 hours . The synthetic netting reduced light intensity (measured as photons) by less than 4% of the ambient light . The time lag between sampling and the beginning of the experiments was about half an hour . At the beginning and end of experiments a subsample of 100 ml from each dialysis sac was fixed with Lugol's iodine solution and another subsample of about 25 ml with formalin (final concentration 1 .5%) . Bacteria and HNF concentrations were counted in formalin fixed samples using epifluorescence microscopy and DAPI staining according to Porter & Feig (1980) . Depending on varying cell concentrations between 2 and 10 ml of these samples were filtered through a 0 .2 µm nuclepore filters for epifluorescence counting . Ciliates were counted in 50 ml Lugol-fixed subsamples under an inverted microscope using the UtermOhl (1958) technique . Growth and grazing coefficients were calculated according to Landry & Hasset (1982) assuming exponential population increase . Net growth rate µ is measured in each dilution mixture from the change in population density during the incubation period (t) : µ = In
Nt • 1 t N No
(1)
with No and N, as the initial and final cell concentrations at the beginning and end of incubation, respectively. As the grazing impact is continuously reduced with increasing dilution of natural lake water, net growth rates are linearly related to the dilution factor . In a dilution series linear regression analysis between the dilution factor and the measured net growth rate yields the gross growth rate (k) as the Y-axis intercept and the grazing coefficient (g) as the negative slope of this relationship. Bacterial cell counts were converted to biomass using mean cell volumes for each season (winter, spring, clear-water phase, summer, and autumn) measured by Simon (1987). Bacterial cell volumes in the
1 13 upper 6 m varied between 0 .039 µm3 in the clearwater phase (June) and 0 .057 µm 3 in mid-summer . HNF were classified by their longest linear dimension into three size categories : < 2 µm, 2-5 µm, and 5-10µm. Larger HNF than 10 µm in length and ciliates were found occasionally, but occurred in numbers too low to be quantified . For each HNF size class a mean volume was calculated assuming cell shapes as rotational ellipsoids with circular cross-sections . Ingestion and clearance rates of HNF and ciliates were calculated according to Davis & Sieburth (1984) : Ingestion rate I (Bact . HNF - 'h - ') = g NBa°t.
(2)
NHNF
with the grazing coefficient g (in units h - ') and the average concentrations of HNF (NHNF) and bacteria (NB,,.,J during the experiments, and Clearance rate C (nl HNF - 'h - ') =
I
(3)
NBact.
Results Near-surface water temperatures and water transparency measured as Secchi disc readings during the period of investigation are indicated in Fig . 1 . Decreasing water temperatures from September through December 1986 and increasing vertical mixing led to a deterioration of mean light conditions for phytoplankton in the upper 6 m of the water column . With decreasing phytoplankton biomass water transparency continuously increased until mid-December . However, in February 1987, when no experiments were conducted, transparency was even higher (13.7 m on 23 February). The increase in water temperature at the onset of stratification and the concomitant decrease in visibility in mid-April mark the beginning of the spring phytoplankton bloom . The phytoplankton biomass peak was reached on 27 April, when the average chlorophyll concentration in the upper 5 m was 75 .5 ug chl .a 1 - ' .
The corresponding phytoplankton biovolume was 4283 mm 3 m -3 (Braunwarth & Tilzer, unpubl . results) . The clear-water phase characterized by low phytoplankton biomass is obvious in Fig. 1 from the end of June through mid-July, when the transparency was more than 5 m . Yet, probably due to the unusually bad weather conditions the clear-water phase was less expressed than in former years when transparency increased from 1 to 10 m within a few days (Lampert, 1978 ; Geller, 1980) . Cell numbers of pelagic bacteria and heterotrophic nanoflagellates (HNF) are given in Fig . 2 . Mean bacterial concentrations in the upper 6 m of the water column varied between 0 .60 x 10 6 cells ml - ' (20 October 1986) and 6 .52 x 10 6 cells ml - ' (29 June 1987) . HNF abundance was lowest in April (0 .54 x 10 3 cells ml - ' on 6 April 1987) and highest in early summer (8 .14 x 10 3 cells ml - ' on 1 June 1987) . From September through December 1986 bacteria and HNF populations varied inversely . In spring 1987 bacterial concentrations closely followed phytoplankton biomass, whereas HNF numbers started to increase with a time lag of about two weeks . A second peak of bacterial abundance was reached in early July after a previous decline of HNF abundance . This pattern of a typical predatorprey relationship is more obvious if one considers biomass instead of cell numbers (Fig . 3) . Through most of the year HNF biomass is a smaller fraction, about 10 and 20%, of bacterial biomass . In late spring and early summer, however, the biovolume of HNF reaches that of free-living bacteria . The grazing pressure on pelagic bacteria is shown in Fig . 4 . In autumn 1986, decreasing grazing pressure coincided with an increase of bacterial cell numbers and biomass (Figs . 2, 3) . In 1987, the changes in bacterial population grazing rates roughly corresponded with varying bacterial biomass . The low grazing rates measured in June coincided with decreasing HNF populations . HNF seem to be the major bacterivores in Lake Constance, since the ingestion rate of HNF closely paralleled grazing pressure on bacteria (Fig . 5) . Here and in the following calculation of HNF clearance rates I assumed that HNF were the sole
114 25
25 Mean water temperature in the upper 1m [ICI Secchi disk transparency [m]
20-
-20
15-
-15
-10
10-
-IRV A A A
5-
0
1 0 I N I D I M I A I M I J I J I 1987 1986
-5
A
0
Fig. 1 . Near-surface water temperature and water transparency at the sampling location .
Bacteria 10 6 cells
12-
HNF
ml -1
10 3 cells ml -1
10-
V C d -0 7
4-
d
C O X:
2
A
A 0
S 1986
1987
Fig. 2 . Mean concentrations of free-living bacteria and heterotrophic nanoflagellates (HNF) in the upper 6 m of the water
column .
115
Bacterial biomass HNF biomass
Fig. 3 . Mean biomass (given as biovolume) of the pelagic bacteria and of heterotrophic nanoflagellates (HNF) in the upper 6 m of the water column.
It
I
vO
0
S I
0 I N I D I I 1986
M I
A I
M I 1987
J I
J I
Fig. 4 . Bacterial population grazing rates obtained from experiments in situ .
A
116
t
147.4
100L LL
Z S d a) a d m L L_
Z S
50-
0
a d L
C 0
aN CC
I
M
I
A
I
M
I
J
1986
1987 Fig. 5 . Ingestion rates of heterotrophic nanoflagellates (HNF) calculated from experiments .
bacterial predators during the experiments . The results must therefore be regarded as maximum estimates . Typically HNF ingest between 10 and 100 bacteria per hour . Apparently low values were obtained in May and June . Considering HNF clearance rates (Fig . 6) the conclusion that HNF become food limited at the height of their population standing stock is even more striking . In June clearance rates were almost zero . The recovery of HNF populations in July and August (Figs . 2, 3) was paralleled by a moderate increase in per capita ingestion and clearance rates . So far only HNF were identified as the major bacterial consumers . Other potential bacterial feeders are either of negligible importance as in the case of ciliates (discussed below) or were excluded from the experiments by the prefiltration of the water used for incubations (cladocerans and copepods) . Ciliates nevertheless strongly affected population dynamics on HNF. As larger zooplankton was excluded from the experiments and other microzooplankton such as rotifers and
developmental stages of crustacea was of minor importance, the grazing pressure on HNF (Fig . 7) has to be primarily caused by ciliate feeding . The first grazing peak in April obviously prevented the rapid increase in HNF standing stocks parallel to that of bacteria, because HNF production had started to increase simultaneously with bacterial production . The abundance of nano- and microciliates in the upper 6 m of the water column is given in Fig. 8 . It is remarkable that ciliates reached their first spring peak simultaneously with phytoplankton and bacteria, but two weeks earlier than HNF. The decline of ciliate concentrations from the end of April through mid-May was the period of maximum increase of HNF biomass . These findings were confirmed during a detailed study of the phytoplankton spring bloom and the response of the microbial loop conducted in 1988 (Weisse et al., submitted). Yet, later in summer the course of population changes of ciliates and of HNF were positively related . If ciliates would primarily feed upon HNF during
1 17
L Z 100S
c LL Z S -4-0
S I
0 I
I D I I
N
M I
A I
M
1986
1987
Fig . 6 . Clearance rates of heterotrophic nanoflagellates (HNF) calculated from experiments .
4
1
-r0
f
al
A-t
0O
S
1
0 I 1986
N
1 0 I
l
M I A
I M I J I J I
A
1987
Fig . 7 . Grazing pressure on heterotrophic nanoflagellates (HNF) measured from in situ experiments .
1 18
25-
20 -
15-
T
10-
5-
0-
S 1
0 1
N
I
D I M
I A I M
I J
I
J
I A
1986 1987 Fig. 8. Mean abundance of nano- and micro-ciliates in the upper 6 m of the water column .
50
536
40-
s Li Z
30-
N
a)
d V
4CU O
20-
d
L
C 0 N N o, C
10-
S I 0 1 N 1 D I
1986
1987
Fig . 9 . Ingestion and clearance rates of ciliates calculated from in situ experiments .
1 19 this period HNF biomass should have collapsed instead of slowly recovering . Ingestion and clearance rates of ciliates reported in Fig . 9 may clarify the trophic relationships between HNF and ciliates . If HNF were the sole food item of ciliates those would have exerted heavy grazing pressure on HNF at the onset of the spring phytoplankton bloom when the build-up of high standing stocks of HNF was retarded . Thereafter, rapidly decreasing grazing pressure through ciliates enabled the mentioned increase in HNF cell number and biomass . From Fig . 9 it is also apparent that ciliates would have starved in July and August if no other food sources than HNF were available . The increasing ciliate populations after the clearwater phase suggest that ciliates switched to a phytoplankton diet . The decreasing ciliate grazing pressure was accompanied by a shift in ciliate species composition from spring through summer (Miller, 1989) . As a consequence ciliate grazing pressure on HNF was reduced thus allowing a recovery of HNF populations . This interpretation is in good agreement with measured grazing rates on HNF (cf. Fig . 7) . As it is not known to what extent phytoplankton was grazed by ciliates during the spring bloom, the calculated clearance and ingestion rates reported in Fig . 9 have to be regarded as minimum values .
Discussion Results presented in this paper were obtained from experiments in situ using a lake water dilution technique . This technique has been first proposed by Landry & Hasset (1982), who measured the grazing impact of marine microzooplankton on phytoplankton assemblages in coastal waters off Washington, USA. Since then it has been used several times in marine studies (Landry et al., 1984 ; Campbell & Carpenter, 1986 ; Verity, 1986a, b) . Three basic assumptions are involved : (1) linear relationship between the dilution factor and grazing, (2) constant gross growth independent from the dilution factor, and (3) exponential cell growth . The first two assumptions are the most critical ones . Feeding is only linearly related
to the probability of encounter between predator and prey as long as neither a lower threshold concentration is transgressed nor substrate concentration is saturating . In the first case feeding ceases and calculated mean ingestion rates are underestimated ; in the second case further increase in substrate concentration is not followed by increased ingestion rates . Thus, if two dilution mixtures are in the range of substrate saturation, no grazing effect is measurable at all or calculated grazing rates are gross underestimations . Consequently, the dilution mixtures have to be chosen in a way that prey concentration is neither too high nor too low . The optimal dilution ratios were tested in preliminary experiments conducted in August and September 1986 (Weisse, unpubl .) . In these experiments dilutions beyond a ratio of 1 : 3 natural to filtered lake water sometimes revealed no further increase in net growth rates of bacteria compared to less diluted mixtures . Thus, the existence of a lower feeding threshold for HNF grazing on bacteria at a bacterial concentration of about 0 .8 x 106 cell ml - ' is possible in Lake Constance in summer. Fenchel (1982) reported a feeding threshold at concentrations of 1 x 10 6 cell ml - ' . Yet, the feeding threshold itself might depend on concentration . In oligotrophic waters of the Red Sea where near-surface bacterial concentrations are in the range of 0 .5-0 .9 x 10 6 cells ml - ' I found some indication for the existence of a feeding threshold when bacterial abundance fell below 0 .6 x 10 6 cells ml - ' (Weisse, 1989) . Seasonal variation of the level of the feeding threshold might explain why grazing rates were indeed linearly related to the dilution factor even in March and early April, when bacterial concentrations were considerably below 1 x 10 6 cells ml - ' in the most diluted dialysis sacs . The second assumption, that gross growth is constant in all dilution mixtures, is best accomplished using dialysis sacs and in-situ incubation as substrates can cross the dialysis membrane (Verity, 1986a) . Furthermore, the natural light climate was not strongly altered by the experimental set-up used in the present study . Dialysis sacs have meanwhile been used successfully in several studies on growth and grazing rates of natural
1 20 pico-, nano-, and micro-plankton assemblages (Landry & Hasset, 1982 ; Landry et al., 1984 ; Verity, 1986a, b) . Ingestion and clearance rates reported in this study are well within the range of published data . Feeding of HNF measured elsewhere with different methods and various species usually yielded ingestion rates between 10 and 80 bacteria consumed HNF - ' h - ' and clearance rates between 0.2 and 79 nl HNF - ' h - ' (reviewed by B . F . Sherr et al., 1986) . In a study conducted parallel to the present in 'Obersee', the main basin of Lake Constance, Jiirgens & Glide (1990) measured ingestion rates ranging from 4-34 bacteria HNF - ' h' depending on the method used. Therefore, the conclusion seems to be justified that HNF in Lake Constance can thrive solely on a bacterial diet . This interpretation is further supported by the fact that HNF production (in terms of biovolume) amounts to about 15 % of bacterial production on the annual average . If we assume a gross growth efficiency of 30 % (Fenchel, 1982 ; Sherr et al., 1983 ; Sherr & Sherr, 1984 ; Landry et al., 1984) HNF would consume about 50 % of the annual bacterial production in Lake Constance . However, in terms of carbon biomass the cropping of bacterial production by grazing flagellates would be somewhat lower as the weightspecific carbon content of bacteria is obviously higher than that of protozoa (Simon & Azam, 1988) . Similar consumption values have been found in comparable marine studies . Sherr et al. (1984) calculated that HNF in Georgia estuaries and offshore waters grazed 30 to 50% of daily bacterial production . Landry et al. (1984) reported that although HNF were the dominant bacterivores in shallow waters off Hawaii maintenance of relatively stable bacterial concentrations could not be attributed solely to grazing by HNF . Thus, although grazing by HNF is the main loss factor of bacterial production in Lake Constance as has already been assumed from indirect evidence by Simon (1987) and Glide (1986) other loss processes such as consumption by larger zooplankton, sedimentation, or autolytic cell disintegration are together of equal importance as HNF grazing . As stated by Glide (1986,
1988) crustacean zooplankters are not important grazers of bacteria in Lake Constance . The two dominant cladoceran species in Lake Constance, Daphnia galeata and D. hyalina have been classified as low efficiency bacterial feeders (Geller & Miller, 1981 ; Gophen & Geller, 1984 ; Gilde, 1988). In the trophogenic zone ciliates are also of minor importance as bacterial consumers . The predominant ciliate species occurring in the upper 20 m of the water column are known as nonbacterivores or low-efficiency bacteria feeders . In the deeper water the fraction of high-efficiency bacteria feeders among the ciliate community might be higher (Weisse & Miiller, 1989 ; Miiller et al., 1990). As larger zooplankton was excluded from the dilution experiments, ciliates were the top predators within the microbial plankton assemblage . In previous studies it has been shown experimentally that ciliates feed upon flagellates (Sheldon et al., 1986 ; Verity & Villareal, 1986 and references therein) . On the assumption that ciliates ingested no other food than HNF clearance rates between 0.2 and 53 .6 pl ciliate - ' h - ' were calculated . This agrees well with previous estimates . Ciliates clearance rates reported in literature generally vary between 0 .1 and 85 pl ciliate - ' h - ' (Spittler, 1973 ; Heinbokel, 1987a, b ; Fenchel, 1980 ; Capriulo, 1982 ; Rivier et al., 1985 ; Verity, 1985) . The measured community grazing rate on HNF amounted to 1600 HNF ml -' d -' . The calculated HNF production rate is in the same order of magnitude (Weisse, unpublished) . Therefore, it is concluded that ciliates largely control HNF population dynamics in Lake Constance throughout most of the year . This might not be true in summer, when grazing pressure on HNF declined although ciliate populations increased, because ciliates used other food during that time. Grazing and clearance rates of HNF and ciliates have been calculated on simplified assumptions . Other food sources such as autotrophic picoplankton, which occurs in typical concentrations of 10 3 to 10 5 cell ml - ' (Weisse, 1988 ; Weisse & Schweizer, 1990), nanophytoplankton, detritus, and attached bacteria have to be considered as potential food for HNF and ciliates .
121 forschungsbereich 248 `Stoffhaushalt des Bodensees' . The technical assistance of G . Baldringer is grateful acknowledged . W . Geller and H . Mtlller provided helpful comments on the manuscript .
References
HNF Heterotr. Nanofl APP Autotr . Picopl .
The microbial loop in Lake Constance
Fig. 10 . The microbial loop in Lake Constance . Known fluxes of organic matter are indicated by solid arrows, hypothetical fluxes by dashed lines . The size range of the main categories is shown at the left margin .
Several ciliate species have been found in Lake Constance which are well known as carnivores . Some are large forms > 200 µm, but others belong to the microplankton (Weisse & Miiller, 1989) . It is therefore assumed that group-cannibalism also occurs among the ciliate community . However, no estimates can be made up to date as to what extent ciliates feed upon ciliates . The same holds true for the HNF assemblage . Our present knowledge of the microbial loop in Lake Constance is summarized in Fig . 10 . Within the microbial loop a linear food chain consisting of bacteria-HNF-ciliates is a potential major pathway transferring bacterial production to higher levels of the planktonic food web . However, it remains a goal for future research whether HNF and ciliate production represent a significant pathway for organic carbon transfer from bacteria to larger zooplankton, or conversely, whether the microbial loop is a sink and dead end of the energy flow within the planktonic system .
Acknowledgements The study was supported by the Deutsche Forschungsgemeinschaft within the Sonder-
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